2632 |Chem. Commun., 2016, 52, 2632--2635 This journal is ©The Royal Society of Chemistry 2016
Cite this: Chem. Commun., 2016,
52,2632
Reactivation from the Ni–B state in [NiFe]
hydrogenase of Ralstonia eutropha is controlled
by reduction of the superoxidised proximal
cluster†
Valentin Radu,
a
Stefan Frielingsdorf,
b
Oliver Lenz
b
and Lars J. C. Jeuken*
a
The tolerance towards oxic conditions of O
2
-tolerant [NiFe] hydro-
genases has been attributed to an unusual [4Fe–3S] cluster that lies
proximal to the [NiFe] active site. Upon exposure to oxygen, this
cluster converts to a superoxidised (5+) state, which is believed to
secure the formation of the so-called Ni–B state that is rapidly
reactivated under reducing conditions. Here, the reductive reactivation
of the membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia
eutropha in a native-like lipid membrane was characterised and
compared to a variant that instead carries a typical [4Fe–4S] proximal
cluster. Reactivation from the Ni–B state was faster in the [4Fe–4S]
variant, suggesting that the reactivation rate in MBH is limited by the
reduction of the superoxidised [4Fe–3S] cluster. We propose that the
[4Fe–3S] cluster plays a major role in protecting MBH by blocking the
reversal of electron transfer to the [NiFe] active site, which would
produce damaging radical oxygen species.
Hydrogenases are metalloenzymes that catalyze the inter-
conversion between H
2
and H
+
s. Their high activities coupled
with high affinities for H
2
have attracted interest in their reaction
mechanism.
1–3
Based on the metal content of the active site,
hydrogenases are grouped into two main classes: [NiFe] and
[FeFe] hydrogenases.
4
Both classes serve as models for designing
sustainable synthetic catalysts for fuel cells and hydrogen
production.
5–9
Maintaining activity in the presence of oxygen is
key for these functions
10
and a sub-class of ‘‘O
2
-tolerant’’ [NiFe]
hydrogenases have therefore been of particular interest.
11,12
‘‘O
2
-tolerant’’ [NiFe] hydrogenases can be clearly differentiated
from the so-called ‘‘standard’’ [NiFe] hydrogenases which,
like [FeFe] hydrogenases, are instantly and almost completely
inactivated by O
2
. Among the most studied O
2
-tolerant [NiFe]
hydrogenases are the membrane-bound hydrogenases (MBHs)
from Ralstonia eutropha,Ralstonia metallidurans,Aquifex aeolicus,
Hydrogenovibrio marinus,andEscherichia coli.
[NiFe] hydrogenases are composed of at least one large
subunit (a) and a small subunit (b).
1,4
The active site has the
same configuration in all [NiFe] hydrogenases and it is deeply
buried into the large subunit (Scheme 1).
When exposed to O
2
, the active sites of standard [NiFe]
hydrogenases are converted to a mixture of inactive states called
‘‘Ni–A’’ and ‘‘Ni–B’’.
13–18
These inactive states are reconverted to the
active Ni–S state in reducing conditions, but the reactivation of the
enzyme from the Ni–A state, also called the ‘‘unready’’ state, can
take hours. In contrast, the Ni–B or ‘‘ready’’ state is reduced to the
active state in a matter of seconds. While the ligand bridging the Fe
and the Ni atom in the Ni–B state has been assigned to an OH
,the
structure of Ni–A remains unclear.
We and others have proposed that an unusual proximal
[4Fe–3S] cluster that is only found in O
2
-tolerant [NiFe] hydro-
genases provides a reductive environment that ensures the
formation of the fast-reactivating Ni–B state.
17,19–22
The tolerance
towards O
2
has also been ascribed to their high affinity for
H
2
, outcompeting and thus excluding O
2
from the active site.
Scheme 1 Schematic representation of the heterotrimeric MBH in the
native cytoplasmic membrane inserted in the quinone-containing tBLM
adsorbed on a SAM. The SAM, adsorbed on a template stripped gold
surface, comprises phase-separated 6-mercapto-1-hexanol spacers
(black) and EO
3
-cholesteryl tethers (gray). The structure of the active site
in the Ni–S state is depicted in the upper-left corner.
a
School of Biomedical Sciences, The Astbury Centre for Structural Molecular
b
Institut fu
¨r Chemie, Sekretariat PC14, Technische Universita
¨t Berlin, Straße des
17. Juni 135, 10623 Berlin, Germany
†Electronic supplementary information (ESI) available: Supporting figures and
experimental details. See DOI: 10.1039/c5cc10382g
Received 18th December 2015,
Accepted 4th January 2016
DOI: 10.1039/c5cc10382g
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The K
M
for H
2
of O
2
-tolerant [NiFe] hydrogenases can be three
orders of magnitude smaller than the O
2
inhibition constant
KO2
i
.
18,20,21
MBHs contain a membrane-integral subunit, cytochrome b,
which couples H
2
oxidation activity to the reduction of the
quinone pool.
12,23–28
The cytochrome bsubunit also anchors
the protein complex to the cytoplasmic membrane, in which
MBH is known to form oligomers (for simplicity, the higher
oligomeric state is not shown in Scheme 1). A distal [4Fe–4S]
cluster of MBH is located close to the surface of the small
subunit and it has been proposed that this enables intermolecular
electron transfer within the dipartite or tripartite supercomplex in
the membrane.
23,26
Electrochemical techniques have been very useful for testing
the catalytic properties of hydrogenases via mediated or direct
electron transfer. Protein film electrochemistry (PFE) has been
especially helpful in elucidating inhibition mechanisms. PFE
monitors the catalytic turnover of an enzyme directly adsorbed
on the electrode while the redox state of the enzyme is controlled
via the applied potential. PFE studies have shown that under H
2
,
O
2
-tolerant [NiFe] hydrogenases recover activity quickly after
being exposed to oxidizing potentials (anaerobic inactivation) or
O
2
.
18–21,29–32
So far, most PFE studies have employed the hetero-
dimeric hydrogenase module only, which is a water soluble
heterodimeric (ab) sub-complex of the MBHs. We have recently
shown that the full heterotrimeric membrane-bound [NiFe]
hydrogenase (MBH) from R. eutropha in equilibrium with the
quinone pool displays enhanced tolerance to oxidizing conditions
compared to the heterodimeric sub-complex.
33
For instance, no
inactivation was observed in the presence of H
2
under (anaerobic)
oxidative redox conditions where PFE of the hydrogenase module
showed Ni–B formation.
In our system, the MBH from R. eutropha, along with its
native lipid environment in the cytoplasmic membrane, is
tethered to the electrode via a mixed self-assembled monolayer
(SAM) (Scheme 1) containing a cholesterol-based tether
(EO
3
-cholesteryl). The tethers are mixed with 6-mercaptohexanol
spacer molecules, which phase-separate on the surface, leaving
space for transmembrane proteins such as MBH. A tethered
bilayer lipid membrane (tBLM), containing E. coli polar lipids,
quinones, and cytoplasmic membrane extracts of R. eutropha,is
then formed on top of the SAM by self assembly. In this system,
the redox-active MBH is in equilibrium with quinones added in
the tBLM, either ubiquinone-10 or menaquinone-7. The redox
state of the quinone pool is controlled via the electrode potential.
In the absence of H
2
, MBH is known to reside in the Ni–B
state, which is thought to protect the active site from irreversible
damage.
19
To confirm that the Ni–B state is formed in MBH in
our setup, multiple H
2
-saturated aliquots of buffer were injected
under anaerobic conditions while the electrode potential was
poised at 0.5 V vs. SHE (Fig. 1, black trace). The transient exposure
leads to the peak-shaped signals shown in the chronoamperograms
in Fig. 1. Upon injection of hydrogen, MBH reactivates, leading to
a rise in oxidative current. As the concentration of the injected gas
decays exponentially with time (while the electrochemical cell is
stirred and flushed with another gas, like N
2
), the oxidative current
returns to the baseline as H
2
is flushed out.
34
The current increase
and decay reveal information about the (re)activation and
inactivation kinetics, respectively. We note that the enhanced
oxygen tolerance of the full heterotrimeric MBH in our system
enables, for the first time, to detect the reactivation under
oxidative conditions, whereas in previous PFE experiments this
was not possible as reactivation required reductive (or mild
oxidative) conditions.
In the case of MBH
wt
donating electrons to ubiquinone,
reactivation after the second injection was 1.5 times faster
compared to the first reactivation based on the slope of the
linear domain of the current increase at a potential of 0.5 V vs.
SHE (normalised rate at 1st reactivation: 92.2 (13.9) 10
4
s
1
;
2nd reactivation: 136.6 (11.5) 10
4
s
1
;n=9;thetimebetween
injections was approximately 400 s). Because the H
2
depletion rate
is identical after both injections, the peak height after the second
injection is thus higher (Fig. S1, ESI†).
To further confirm that the inactive state in our setup represents
the Ni–B state, O
2
was injected into the cell solution (via an air-
saturated aliquot of buffer) after two injections of substrate
(Fig. 1, blue trace). The O
2
injection ensures the total conversion
of the enzyme to the Ni–B state.
17,21
AthirdaliquotofH
2
-saturated
buffer was injected into the cell solution after the complete
depletion of O
2
. A control experiment was recorded replacing O
2
with N
2
(Fig.1,blacktrace).ThereactivationkineticsafterO
2
treatment was identical to that after N
2
injection, confirming that
the Ni–B state is indeed responsible for the observed kinetics.
The reactivation rate decreases as the time of the oxidative
poise between the 2nd and 3rd injection increases (Fig. S2,
ESI†). The reactivation rate after the 1st H
2
injection is only
marginally faster than that of the 3rd injection after prolonged
oxidative poise, in line with the expectation that the MBH
mainly resides in the Ni–B state in anaerobic electron-deficient
conditions. The inactivation kinetics indicates that the accumulation
of Ni–B is complete approximately 400 s after all H
2
is removed
from solution.
Having confirmed that inactivation is due to Ni–B formation,
we studied the reactivation kinetics by analysing the slope
of current increase after injection of H
2
. First, the potential
dependence of the reactivation kinetics was investigated using
menaquinone-containing tBLMs (Fig. 2a). Menaquinol has a
Fig. 1 Chronoamperograms showing the evolution of the H
2
oxidation
current of MBH
wt
after H
2
pulses intercalated with one O
2
/N
2
pulse (0.499 V
vs. SHE; ubiquinone-containing tBLMs; 30 1C; pH 7.4; H
2
concentration after
injection: 100 mM; O
2
concentration after injection: 28 mM).
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lower oxidation potential, which enables studies over a wider
potential window (0.2–0.6 V). Reactivation rates were only
marginally slower at higher potential (a 17% decrease of the
slope of the reactivation trace was observed when increasing the
potential from 0.2 V to 0.4 V). A similar potential dependence of
Ni–B reactivation was observed with the hydrophilic heterodimeric
forms of the MBHs from A. aeolicus and E. coli.
31,35
Next, we
studied the temperature dependence and higher temperatures
significantly increased the reactivation rate (Fig. 2b). The
temperature profiles allowed the determination of the activation
energy (E
a
) from Arrhenius plots using the slope of the reactivation
trace as the temperature dependent variable (Fig. 3). E
a
was
determined to be 141.5 (5.0) kJ mol
1
(n= 3) at 0.3 V, in line
with values obtained with Hyd-1 for which E
a
varied from a value of
56.8 kJ mol
1
at 0.035 V to 96.3 kJ mol
1
at 0.235 V.
35
To determine how the reactivation kinetics is dependent on
the unusual [4Fe–3S] promixal cluster, these experiments were
repeated with an MBH variant (MBH
C19G/C120G
). The variant carries
a [4Fe–4S] cluster proximal to the [NiFe] site, thus resembling the
[FeS] cluster configuration of standard O
2
-sensitive [NiFe] hydro-
genases.
32
MBH
C19G/C120G
is known to share similar catalytic
properties with MBH
wt
and the same active site structure as any
[NiFe] hydrogenase.
32
As previously observed for this variant
(Supplementary Table 1 in ref. 32), the activity of MBH
C19G/C120G
was 4 to 5 times lower compared to MBH
wt
in our setup (Fig. 4a),
which is attributed to the lower expression level of the variant, an
unfavourable potential of the engineered [4Fe–4S] cluster and/or a
higher sensitivity towards oxygen, irreversibly deactivating more of
the MBH during extraction from R. eutropha.Importantlyand
unexpectedly, it was found that MBH
C19G/C120G
reactivates 7 times
faster than the MBH
wt
(Fig. 4b). The normalised rate of current
increase for MBH
C19G/C120G
, after H
2
injection, was 1027.7
(41.7) 10
4
s
1
(n= 11). To determine if the faster reactivation
kinetics is due to a lower activation energy, the temperature
dependence of the kinetics was determined. As the variant is less
active, it was not possible to accurately probe the reactivation at
low potentials (the lower currents led to unacceptable signal-to-
noise ratios). It was observed that the variant was also less
thermostable. Nonetheless, in spite of the smaller temperature
window, it is clear that the reactivation energy is significantly
lower (Fig. 3). At 0.5 V, E
a
was 22 8 kJ mol
1
(n= 4), which is
remarkably similar to that determined for H
2
oxidation.
36,37
These results suggest that the reactivation kinetics of MBH
wt
is rate limited by the reduction of the superoxidized [4Fe–3S]
5+
cluster. The relatively slow reduction of the [4Fe–3S]
5+
cluster
compared to that of other [FeS] clusters can be explained by the
chemical reorganisation that is coupled to this step. In contrast
to MBH
wt
, the reactivation kinetics of MBH
C19G/C120G
was independent
of the time of exposure to oxidizing electrode potentials (i.e.,the
inactivation time, Fig. S3, ESI†). We propose that the inactivation
kinetics of MBH
wt
(Fig. S2, ESI†) are determined by the super-
oxidationofthe[4Fe–3S]cluster,whichisabsentinMBH
C19G/C120G
as the standard [4Fe–4S] cluster cannot be superoxidised.
It has been previously observed that standard [NiFe] hydro-
genases like the ones from Allochromatium vinosum and Desul-
fovibrio gigas display a much slower anaerobic inactivation
compared to O
2
-tolerant [NiFe] hydrogenases like the ones
Fig. 2 Chronoamperometric traces recoded after short H
2
pulses showing
the increase of the reactivation rate with decreasing potential (a) and
increasing temperature (b) (menaquinone-containing tBLMs; the traces
are aligned according to the injection point, 30 1C (a); 0.299 V (b); pH
7.4; H
2
concentration after injection: 100 mM; the first pulse is not shown).
Fig. 3 Arrhenius plots for Ni–B reactivation (the slope of the reactivation trace,
m, was taken as the temperature-dependent variable; MBH
wt
:traceswere
recorded at 0.299 V using menaquinone-containing tBLMs; MBH
C19G/C120G
:
traces were recorded at 0.499 V using ubiquinone-containing tBLMs).
Fig. 4 Cyclic voltammograms (a) and chronoamperograms (b) showing
activity levels (a) and reactivation traces (b) for MBH
wt
and MBH
C19G/C120G
(30 1C; pH 7.4). In (a): ubiquinone- and menaquinone-containing tBLMs;
10 mV s
1
. In (b): 0.499 V vs. SHE; ubiquinone-containing tBLMs; H
2
concentration after injection: 100 mM.
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from R. eutropha and A. aeolicus.
19,21
In the case of O
2
-tolerant
hydrogenases, protection of the active site, achieved by Ni–B
formation, is related to the formation of the superoxidised
cluster.
24,35,38
When the reduction of the superoxidised proximal
cluster is slow, as observed here, it follows that the [FeS] relay
cannot function as an efficient electron hub for the active site. We
therefore hypothesize that the ‘‘locking’’ of the proximal cluster in
the superoxidized state prevents the irreversible damage to the
active site under aerobic conditions by prohibiting electron
transfer to the [NiFe] active site forming radical oxygen species.
The temporary shutdown of the electron relay responsible for
disabling the active site reactivity would impede the irreversible
deterioration of the protein and implicitly the complete loss of
enzymatic activity. In contrast, standard [NiFe] hydrogenases
do not shut down the electron relay and continuous electron
transfer to the active site could form damaging radicals under
aerobic conditions, possibly resulting in the formation of the
Ni–A state and other irreversible inactive states.
39
In conclusion, we have confirmed that in the absence of H
2
and under electron-deficient conditions, the full heterotrimeric
MBH in our experimental system forms the Ni–B state, as
previously extensively shown for the hydrogenase module (a
water soluble heterodimeric (ab) sub-complex of the membrane-
bound hydrogenases). The MBH variant carrying a standard
[4Fe–4S] proximal cluster reactivated many times faster than
wild-type MBH, suggesting that the reduction of the superoxidised
proximal cluster determines the kinetics of Ni–B reactivation. Active
site protection in oxidative conditions is proposed to be achieved by
the formation of the superoxidized state of the proximal cluster,
which interrupts the functioning of the [FeS] electron relay and
prevents the formation of radical oxygen species at the [NiFe] site.
The research leading to these results has received funding
from the European Research Council under the European
Union’s Seventh Framework Programme (FP/2007–2013)/ERC
Grant no. 280518 (V. R. and L. J. C. J.) and from the DFG cluster
of Excellence ‘‘Unifying Concepts in Catalysis’’ (S. F. and O. L.).
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